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Genetic Depletion of Amylin/Calcitonin Receptors Improves Memory and Learning in Transgenic Alzheimer’S Disease Mouse Models

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Genetic Depletion of Amylin/Calcitonin Receptors Improves Memory and Learning in Transgenic Alzheimer’S Disease Mouse Models Genetic Depletion of Amylin/Calcitonin Receptors Improves Memory and Learning in Transgenic Alzheimer’s Disease Mouse Models. Aarti Patel University of Alberta Hospital Ryoichi Kimura Sanyo-Onoda City University: Sanyoonoda Shiritsu Yamaguchi Tokyo Rika Daigaku Wen Fu University of Alberta Rania Soudy University of Alberta David MacTavish University of Alberta David Westaway University of Alberta Jing Yang University of Alberta Rachel Davey University of Melbourne Jeffrey Zajac University of Melbourne Jack H. Jhamandas ( [email protected] ) University of Alberta https://orcid.org/0000-0002-4688-6500 Research Article Keywords: Amylin, amylin receptor, calcitonin receptor, Alzheimer's disease, amyloid-β protein, long term potentiation, hippocampus, spatial memory Posted Date: May 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-515476/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/24 Version of Record: A version of this preprint was published at Molecular Neurobiology on July 27th, 2021. See the published version at https://doi.org/10.1007/s12035-021-02490-y. Page 2/24 Abstract Based upon its interactions with amyloid β peptide (Aβ), the amylin receptor, a Class B G protein-coupled receptor (GPCR), is a potential modulator of Alzheimer’s disease (AD) pathogenesis. However, past pharmacological approaches have failed to resolve whether activation or blockade of this receptor would have greater therapeutic benet. To address this issue, we generated compound mice expressing a human amyloid precursor protein gene with familial AD mutations in combination with deciency of amylin receptors produced by hemizygosity for the critical calcitonin receptor subunit of this heterodimeric GPCR. These compound transgenic AD mice demonstrated attenuated responses to human amylin- and Aβ-induced depression of hippocampal long term potentiation (LTP) in keeping with the genetic depletion of amylin receptors. Both the LTP responses and spatial memory (as measured with Morris Water Maze) in these mice were improved compared to AD mouse controls and, importantly, a reduction in both the amyloid plaque burden and markers of neuroinammation was observed. Our data support the notion of further development of antagonists of the amylin receptor as AD-modifying therapies. Introduction A dening pathological feature of Alzheimer’s disease (AD) is the presence of soluble oligomers of amyloid beta (Aβ) that aggregate into extracellular brillary deposits known as amyloid β-plaques (1). Although the role of the brain Aβ in AD is not completely resolved, available evidence indicates that it may initiate and/or contribute to the process of neurodegeneration (2, 3). Neuronal dysfunction, synaptic disruption, neuroinammation and vasculopathy are key perturbations of AD pathology and each is linked to the presence of Aβ in the brain (1, 3). Aβ protein has been shown to interact with a number of G protein coupled receptors (GPCRs) to modulate major cognitive and pathological features of AD and have thus become attractive therapeutic targets for the development of disease modifying treatments for AD. We have shown that human amylin, a 37-amino acid peptide identied in pancreas of diabetics, causes neurotoxicity in a manner very similar to Aβ, and that the effects of the human amylin and Aβ on neurons appear to be expressed via the same receptor, the amylin receptor, a Class B G protein-coupled receptor (4). Pharmacological studies using the amylin receptor antagonist, AC253, have shown benecial effects on synaptic function, spatial memory and learning and neuroinammation in transgenic mouse models of AD (5, 6, 7). However, other studies have reported that the systemic administration of human amylin or pramlintide, a synthetic non-amyloidogenic analog of amylin, is capable of improving decits in spatial memory (8, 9). Thus, to address these contradictory ndings arising from the pharmacological evidence, we generated a line of transgenic (Tg) AD mice expressing a genetically-induced decit in amylin/calcitonin receptors, but carrying the full amyloid protein precursor (APP) gene and resultant over- expression of Aβ. If the amylin receptor is indeed a mediator of deleterious effects of Aβ, one would expect a signicant reduction and/or delay in the development of age-dependent pathology and memory- related decits in the AD mice bearing a reduced complement of amylin receptors compared to AD mice. Page 3/24 Dimerization of the calcitonin receptor (CTR) with one of the three receptor activity-modifying proteins (RAMP1, RAMP2 or RAMP3) yields subtypes of amylin receptors (AMY1-3) that bind amylin with varying anities (10, 11). As such, CTR is a critical component for the functionality of the amylin receptor, i.e. without CTR there is no functional amylin receptor even if RAMPs are present. We have previously shown that in cell cultures of human fetal neurons, siRNA downregulation of CTR blunts Aβ-induced cell death (4). Thus, to extend these in vitro ndings, we sought to genetically deplete CTR receptors in the transgenic AD mice. Since homozygosity for CTR null alleles is embryonic lethal, we sought to assess the impact of hemizygosity for the CTR locus. We thus obtained stocks of CTR mice with one inactivated CTR allele (HetCTR) (12). These HetCTR mice, producing 50% of the amylin receptor complement of wild- type (WT) mice, were crossed with two strains of transgenic AD mice. We used TgCRND8 and 5xFAD lines of mice, both of which express APP695 and develop Aβ-related neuropathology consisting of large numbers of diffuse plaque amyloid deposits in tandem with a progressive deterioration of spatial memory (13, 14). Crossing such hemizygous null CTR (HetCTR) mice with TgCRND8 or 5xFAD mice produces HetCTR + TgCRND8 or 5xFAD compound Tg mouse offspring, which have 50% depletion of amylin receptors but will continue to over-express Aβ deriving from the separate APP695 transgene array. Thus, the compound mice could be used to test whether or not a constitutive genetic depletion of amylin receptors alters the AD-related pathology and memory-related decits that follow from expression of FAD alleles of human APP695. More generally, this approach allows a critical assessment of some contradictory pharmacological ndings and the potential benets of modulating the AMY/CTR axis in clinical dementia settings. Materials And Methods All experiments were conducted in compliance with the guidelines set by the Canadian Council for Animal Care and with the approval of the Human Research Ethics Board and Animal Care Use Committee (Biomedical Sciences) at the University of Alberta (Protocol AUP00000268). Chemical and Reagents: All commercially available chemicals were of analytical grade and used without further purication. Oligomeric Aβ1−42 was prepared according to published protocol (15). Briey, Aβ1−42 (rPeptide) was dissolved to 1 mM in 100% hexauoroisopropanol, hexauoroisopropanol was removed under vacuum, and the peptide was stored at -20°C. For oligomeric conditions, the peptide was rst re-suspended in DMSO to 5 mM, then water added to bring it to a nal concentration of 1 mM, and the peptide incubated at 4°C for 24 h. The hAmylin (AnaSpec Inc.) was dissolved to 1 mM in water and kept at -80°C. Aliquots of Aβ and hAmylin, were further diluted to nal application concentration with cell culture medium. Animal models: Mice were maintained in standard laboratory housing conditions (20 ± 1°C; 70% ± 10% humidity; 12:12 h light/dark cycle). Access to standard rodent chow and water was available ad libitum. Page 4/24 For behavior and histological studies, we used non-Tg (C57BL/6xC3H background) and TgCRND8 mice (human APP695 transgene array incorporating Swedish K670M/N671L and Indiana V717F mutations superimposed upon a C57BL6xC3H genetic background), that have been described previously to exhibit Aβ plaques and cognitive decits from 3 months of age onwards (13); these mice originating from the University of Toronto are being deposited at the Jackson Lab, Bar Harbor ME (JAX # 020661). 5xFAD (amyloid precursor protein (APP)/Presenilin 1(PS1) double transgenic mice with ve familial AD mutations (14) mouse breeding stocks were obtained from the Jackson Laboratory (JAX #006554) and correspond to a C57BL6xSJL hybrid background. Additionally, heterozygous CTR (HetCTR) age-matched mice (C57BL/6J background) with a 50% depletion of CTR expression were obtained using breeding pairs provided from Drs. RA Davey and JD Zajac (University of Melbourne, Australia), these being obtained as C57/BL6 congenic stock15. Crosses of HetCTR and heterozygous Tg-positive mice (5xFAD or TgCRND8) were set to obtain compound heterozygotes (i.e. hemizygous for a functional CTR locus and heterozygous for carrying the APP-expressing transgene array). Both male and female mice were used in our study. Early and late stage male and female AD mice with and without CTR depletion and their WT littermate controls were evaluated at approximately 4 and 8–12 months of age, respectively and described henceforth as 4 and 8 months of age. In both cases, the animals were stratied by age to maintain equivalent age distributions between WT and transgenic experimental groups. Genotyping mouse lines: TgCRND8 mice were genotyped using PCR primers in the human APP coding region and the hamster PrP 3' untranslated region, 5'.TGTCCAAGATGCAGCAGAACGGCTACGAAAA.3' and 5’.AGAAATGAAGAAACGCCAAGCGCCGTGACT.3’. PCR with Taq polymerase (Invitrogen) was performed with a 94 °C melt step (180 sec) followed by 35 cycles using a 94 °C melt temperature (20 sec), hybridization at 68 °C (20 sec) and extension at 72 °C (90 sec). 5xFAD mice were genotyped with APP and Presenilin 1 primer sets. APP 5'.AGGACTGACCACTCGACCA.3' transgene Forward and 5'.CGGGGGTCTAGTTCTGCA T.3' - transgene Reverse, with 5'.CTAGGCCACAGAATT GAAAGA TCT.3' Internal Positive Control Forward and 5'.GTAGGTGGAAATTCTAGCATCATC. 3’ Internal Positive Control Reverse primer. The APP amplicon is 377 bp, the control amplicon 324 bp. Cycles were 35 cycles using a 94 °C melt temperature (30 sec), hybridization at 51 °C (60 sec) and extension at 72 °C (60 sec).
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